The controlled degradation of most cellular proteins is performed by large protein complexes known as proteasomes that are found in both the nucleus and cytoplasm of an cells. Proteasome activity is central to almost all cell processes, including aspects of protein synthesis and turnover, cell cycle control, metabolic response to environment and immune response, and is implicated in a number of disease states, including cancer and Alzheimer's disease. While the catalytic mechanism of the proteolytic sites has been clarified by recent structural work, there is only a preliminary upstanding of the factors involved in regulation and targeting of proteasome activity. This proposal seeks to determine the interactions of the proteasome with three of the best-characterized regulators of its activity. The first of these is the major activator, PA700, which is responsible for targeting the proteasome to ubiquitinated substrates. Hydrolysis of this class of substrates is believed to be the single most important cellular role of the proteasome. Using electron of negatively stained specimens, we have shown that PA700 binds to the 20S proteasome and converts in to another previously-isolated form (26), in an apparently cooperative manner. Moreover, another protein complex that increases the activation of proteasome by PA700, called modulator, promotes the formation of the PA700 caps without itself forming any stable association with the resulting 26S proteasome. They now seek to correlate the observed structural modification with proteolytic activity, and to better define the organization of the multiple subunits and activities of the PA700 proteasome regulator, by extending our electron microscopy and image analysis studies to include cryo-electron microscopy and three-dimensional reconstructions. A second major component of this proposal is the structural examination of an alternative activator of the proteasome. PA28, which is responsible for the role of the proteasome in antigen presentation. They will define its structural organization, and determine its interaction with PA700 in mixed complexes. The third component of this proposal is a parallel study of the binding site and structural correlates of a specific protein inhibitor of the proteasome, P131, and its effects on formation of activated proteasomes.